The norepinephrine transporter regulates dopamine-dependent synaptic plasticity in … · 52 transporters and change the direction of catecholamine flux. Comparably, early studies

1

The norepinephrine transporter regulates 1

dopamine-dependent synaptic plasticity in the 2

mouse dorsal hippocampus 3

Alex Sonneborn1,2,* and Robert W. Greene1,2,* 4

1 Department of Psychiatry, UTSW Medical Center, 5323 Harry Hines Blvd, Dallas, TX, 75205, USA 5 2 Department of Neuroscience, UTSW Medical Center, 5323 Harry Hines Blvd, Dallas, TX, 75205, USA 6 *[email protected]; [email protected] 7 8 Abstract 9

The rodent dorsal hippocampus is essential for episodic memory consolidation, a process 10

dependent on dopamine D1-like receptor activation. It was previously thought that the ventral tegmental 11

area provided the only supply of dopamine to dorsal hippocampus, but several recent studies have 12

established the locus coeruleus (LC) as a second major source. However, the mechanism for LC-13

dependent dopamine release has never been explored. Our data identify norepinephrine transporter 14

reversal as one plausible mechanism by demonstrating that transporter blockade can reduce dopamine-15

dependent long-term potentiation in hippocampal slices. We also suggest that presynaptic NMDA 16

receptors on LC terminals may initiate this norepinephrine transporter reversal. Furthermore, as 17

dopamine and norepinephrine should be co-released from the LC, we show that they act together to 18

enhance synaptic strength. Since LC activity is highly correlated with attentional processes and memory, 19

these experiments provide insight into how selective attention influences memory formation at the 20

synaptic and circuit levels. 21

Introduction 22

Adrenergic signaling in the mammalian brain is largely controlled by a network of remarkably 23

divergent axon projections arising from locus coeruleus (LC) neurons1,2. These LC axons were once 24

thought to exclusively release norepinephrine (NE)3, but recent chemical evidence reveals that their 25

specific activation can also increase extracellular dopamine (DA)4-7. In accordance with this, LC 26

stimulation is sufficient to modulate DA-dependent changes in learning and synaptic physiology within the 27

rodent dorsal hippocampus5,8-10. Dopamine D1-like receptors are abundantly expressed in this region, 28

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2

where they play an essential role in promoting many forms of long-term synaptic potentiation (LTP), 29

especially in area CA111. However, in CA1, projections from canonical DA-releasing nuclei such as the 30

ventral tegmental area (VTA) are sparse compared to those of the LC8,12, indicating that DA receptor 31

activation in this area is mainly due to LC activity. Yet despite data supporting the LC as the main source 32

of DA in dorsal hippocampus, the mechanism underlying its release has never been explored. 33

One hypothesis for the mechanism of LC DA release is by reverse transport through the 34

norepinephrine transporter (NET). Under normal conditions, the NET is responsible for the reuptake of 35

both NE and DA after they are released13,14. In contrast, the presence of amphetamines allows the NET to 36

efflux catecholamines from LC varicosities15, and DA released in this way potentiates synaptic strength in 37

dorsal CA116. Furthermore, the closely related dopamine transporter (DAT) can reverse its flux under 38

more physiological conditions than amphetamine application (for a review, see Leviel (2017))17. These 39

conditions include a rise in intracellular [Na+] and [Ca2+] following action potential firing18,19, activation of 40

NMDA receptors20,21, and phosphorylation by CAMKII or PKC22. Because the amino acid sequences of 41

DAT and NET are almost 80% homologous23, we propose that the NET will also efflux cytosolic DA from 42

LC axons under similar physiological conditions. Below we investigate this possibility in the dorsal 43

hippocampus, where DAT expression is not detectable16,24, by designing a DA-dependent LTP that is 44

significantly attenuated after the NET is blocked. 45

In support of a more detailed model for NET-mediated DA release, an existing theory posits that 46

high-frequency glutamate activity may play a role25. The authors speculate that elevated pyramidal cell 47

firing in response to environmental stimuli can result in glutamate spillover26, leading to activation of 48

presynaptic NMDA receptors on LC terminals and enhanced vesicular NE release. Taking this idea one 49

step further, Olivier et al. discovered that an NMDA-dependent rise in striatal DA is nearly abolished by 50

GBR12909, a selective DAT blocker20. This indicates that NMDA receptors can somehow interact with 51

transporters and change the direction of catecholamine flux. Comparably, early studies in dorsal 52

hippocampus reported increased extracellular NE and DA after NMDA receptor agonist application27,28. 53

To our knowledge, no studies have attempted to associate this DA transmission with NET reversal, or 54

looked at its ability to regulate synaptic plasticity. With this in mind, we deleted NMDA receptors from 55

catecholamine terminals and saw a decrease in DA-dependent LTP in dorsal hippocampus. 56



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Lastly, given that both DA and NE can modulate synaptic plasticity in dorsal CA129, along with the 57

indication of their co-release from the LC, we presume that they are working together to influence 58

synaptic strength in this region30. Our final experiment shows that simultaneous application of DA and NE, 59

but not either of them alone, can strengthen a weaker form of hippocampal LTP. The implications of these 60

results are then discussed in the context of the LC’s purported role in selective attention31, and how 61

glutamate can interact with catecholamines to organize attention-driven memory formation at the synaptic 62

and circuit-specific levels. 63

Results 64

The norepinephrine transporter (NET) contributes to dopamine-dependent potentiation in the 65

dorsal hippocampus 66

If the NET is capable of controlling DA efflux in dorsal hippocampus, then blocking it should 67

attenuate DA-dependent synaptic potentiation. To test this, we developed a strong theta-burst LTP 68

protocol (strLTP) by stimulating CA3 axons and recording the slope of field excitatory postsynaptic 69

potentials (fEPSPs) from stratum radiatum dendrites of CA1 (Fig.1A-D). This protocol was based on 70

previous methods used to generate catecholamine-dependent potentiation in hippocampus32,33. 71

Importantly, our strLTP was not blocked by co-application of β-adrenergic (propranolol) and α1-adrenergic 72

(prazosin) receptor antagonists (Fig. 1E, comparison between strLTP with no-drug from Fig. 1D and 73

strLTP with drug from Fig. 2A). However, following the addition of the D1-like receptor antagonist SCH 74

23390, a robust blockade of LTP occurred over the last 30 minutes of recording (Fig. 2A, red traces), 75

indicating that strLTP is dependent on DA receptors, but not adrenergic receptors. Next, we administered 76

the same strLTP stimulation, but substituted nisoxetine, a NET blocker, for SCH 23390. Treatment with 77

nisoxetine produced a similar reduction in LTP (Fig. 2B, green traces), suggesting that DA signaling in the 78

dorsal hippocampus requires NET activity. Likewise, a genetic deletion of the NET from LC neurons also 79

greatly reduced strLTP amplitude after 1 hour (Supplementary Fig. 1). 80

Blocking α2-adrenergic receptors does not reduce the effect of NET antagonism 81

Because blocking the NET will flood synapses with NE, one possible confound is over-activation 82

of inhibitory α2-adrenergic autoreceptors, leading to a decrease in overall LC excitability and 83



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neurotransmitter release34. This may cause a reduction in LTP based on an indirect decrease in total NE 84

and/or DA levels. To control for this, we repeated the aforementioned experiments with the inclusion of 85

RS 79948, an α2-receptor antagonist, in the bath with propranolol and prazosin. Interestingly, adding RS 86

79948 caused a significant increase in fEPSP slope over the first 30 minutes after strLTP stimulation, but 87

not the last 30 minutes (Fig. 2C, blue traces). This effect could be due to greater vesicular NE release 88

reaching concentrations high enough to displace propranolol and activate β-adrenergic receptors, a 89

process known to enhance early LTP35. In line with our prior results, the further addition of nisoxetine was 90

still able to diminish the magnitude of strLTP over the last 30 minutes (Fig. 2D, green traces), reinforcing 91

the finding that NET may contribute to DA signaling in dorsal hippocampus. 92

NMDA receptor knock-out from catecholamine neurons reduces the magnitude of dopamine-93

dependent LTP 94

Activation of glutamate receptors, in particular NMDA, is capable of enhancing catecholamine 95

release in hippocampus27,28,36. Expanding on the mechanism of DA release from the NET, we asked if 96

presynaptic NMDA receptors on LC terminals were functionally coupled to NET reversal, and thus 97

involved in our NET/DA-dependent LTP. To expand on the mechanism of DA release from the NET, we 98

asked if presynaptic NMDA receptors on LC terminals could functionally be coupled to NET reversal, and 99

thus involved in our NET/DA-dependent LTP. To approach this question, we first confirmed that NMDA 100

receptors co-localized with the norepinephrine transporter on LC axon terminals in the dorsal 101

hippocampus (Supplementary Fig. 2). To our knowledge, this is the first time that co-localization of these 102

proteins has been shown in LC terminals in any area of the brain. 103

We next wanted to check if this co-localization was important for the expression or maintenance 104

of LTP in dorsal CA1. To do this, the NR1 subunit of NMDA receptors had to be genetically deleted from 105

catecholamine neurons, since blocking NMDA receptors would prevent LTP. This was done by crossing a 106

mouse expressing Cre recombinase under the control of the tyrosine hydroxylase (TH) promoter with a 107

floxed NMDA-NR1 subunit mouse. Cre-negative controls for these mice showed normal strLTP (Fig. 3, 108

filled circles), whereas the LC-NR1 knockouts exhibited decreased LTP magnitude throughout the full 109

hour after LTP induction (Fig. 3, open circles). Since dorsal CA1 receives very little VTA input, we 110

interpreted these effects as being predominantly due to NMDA deletion from LC neurons. However, the 111



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results do not rule out possible compensatory effects of NMDA receptor knockout resulting from Cre 112

expression in TH-positive neurons during development37. 113

Concurrent activation of DA and NE receptors is required for LTP enhancement in CA1 114

The regulation of synaptic plasticity by DA or NE is well documented in dorsal hippocampus38,39. 115

For example, D1/D5 receptor antagonists in CA1 can inhibit synaptic potentiation40 and contextual 116

learning41, while agonists of β-adrenergic receptors are sufficient to lower the threshold for LTP 117

initiation42. Yet in dorsal CA1, whether or not coincident activation by both catecholamines is needed to 118

enhance LTP has never been examined. This question remains of great importance, as it has been well 119

established that the LC can release DA and NE together. Accordingly, we developed a weak LTP (wLTP) 120

stimulation paradigm (Fig. 1C&D) and tested if an interaction between DA and NE was necessary to 121

strengthen it. 122

Bath application of NE alone had no effect on the magnitude of wLTP; although a decrease in 123

baseline glutamatergic signaling before wLTP stimulation was apparent (Fig. 4A, 16-30 mins). The latter 124

phenomenon has been recorded previously43, and is likely due to NE activating α1-receptors on 125

interneurons to increase their feed-forward/lateral inhibitory drive44. Surprisingly, washing in DA alone had 126

no effect on either wLTP magnitude or basal glutamate transmission (Fig. 4B). This seems 127

counterintuitive considering that activation of D1-like receptors by selective agonists (e.g. SKF-81297) 128

can reliably evoke LTP in dorsal CA111. However, the result is consistent with multiple reports showing no 129

change in CA1 excitatory transmission in response to bath applied DA45-47. It is unclear why this occurs, 130

but one explanation could be that over activation of inhibitory D2-like receptors negates the excitatory D1-131

like receptor activation in this region. Interestingly, even though neither of the catecholamines in isolation 132

was able to produce stronger LTP, their simultaneous application resulted in a significant increase 133

compared to the control wLTP (Fig. 4C). These findings pair well with the following conclusion that the LC 134

utilizes both DA and NE to optimize memory storage, mainly during periods of enhanced attention to 135

salient stimuli. 136

Discussion 137



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Taken together, these results allude to the LC orchestrating coincident release of NE and DA in 138

the dorsal hippocampus using two separate mechanisms. The first is the widely accepted vesicular 139

release of NE48, and the second is reverse transport of DA from the NET as shown in this study. A reason 140

for separate release mechanisms is still unclear, but one plausible explanation is that they are used to 141

facilitate a molecular link between attention and memory49, especially since the LC is heavily involved in 142

both cognitive processes at the behavioral level50. Below we postulate that their co-release should only 143

occur when an animal devotes sustained attention to salient stimuli associated with prolonged LC firing. If 144

this happens, NE and DA can interact in CA1 to help exclusively potentiate the most relevant synapses 145

for future memory consolidation (Fig. 4). 146

When an animal is awake but not experiencing anything particularly interesting in its environment, 147

the LC releases a low, background level of NE from vesicles using a slower, tonic firing pattern (~3 Hz). In 148

the hippocampus, this leads to a general suppression of activity by activating higher affinity α1-adrenergic 149

receptors on interneurons in the area43,44. During times of elevated arousal and selective attention to 150

salient stimuli, higher frequency (~16 Hz) phasic activity51 transiently boosts extracellular levels of NE 151

34,52. These quick increases in NE are theorized to recruit lower affinity β-adrenergic receptors on 152

hippocampal pyramidal cells to amplify more active glutamatergic inputs, while the α1-receptors continue 153

to reduce the noise generated by less active ones53. Therefore, NE helps the most immediately relevant 154

and strongest signals prevail over those that are firing slower and likely carrying less important 155

information. Elaborating on this, Mather, et al. 25 propose that very active glutamatergic synapses can in 156

turn augment the release of NE after glutamate spillover activates presynaptic NMDA receptors on 157

nearby LC axons. This would create a local positive feedback loop between the most rapidly firing 158

glutamatergic input and phasically active LC terminals, with less active circuits remaining suppressed as 159

they are unable to trigger this positive feedback. In the hippocampus, this system presumably optimizes 160

circuit organization to reduce the overlap between stored memory traces. 161

Our data expand on these theories and suggest that presynaptic LC-NMDA receptors can 162

similarly initiate DA signaling from LC axons in dorsal hippocampus, since their deletion weakens DA-163

dependent LTP (Fig. 3). This effect makes sense within the framework of attention being a driving force 164

for memory formation49. For instance, when strong glutamatergic signaling in response to salient 165



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environmental cues couples with phasic LC firing in CA1, excess glutamate can overflow from the 166

synapse and bind to NMDA receptors on LC terminals25. At the same time, salience-guided, phasic action 167

potential firing in LC terminals will influx Na+ and Ca2+, removing the Mg2+ block and allowing even more 168

cation influx through NMDA receptors. Calcium entering the neuron via this process may then promote 169

the function of CAMKII or PKC, kinases capable of interacting with54 and phosphorylating transporters to 170

reverse their direction55. Likewise, since monoamine transporters are known to move neurotransmitters 171

using the energy stored in Na+ gradients56, a switch to higher intracellular Na+ during action potential 172

bursting could alter the ionic equilibrium enough to move DA out through the NET. In favor of this idea, it 173

is known that the NET can reuptake DA nearly as well as NE in the hippocampus under normal 174

conditions14, hinting that the reverse mechanism may be possible. Figure 2 explores this idea and 175

highlights that DA released in this way seems to be physiologically relevant, as blocking or deleting the 176

NET is capable of reducing DA-dependent LTP. 177

Functionally, this non-canonical DA efflux likely arose as a form of coincidence detection in dorsal 178

CA1. Here it will potentiate only the most prevalent glutamatergic inputs that were selected by the 179

preceding NE modulation of glutamatergic attentional resources. In other words, once a stimulus 180

becomes salient enough to outcompete the background noise, DA is released and interacts with NE to 181

enhance synaptic strength (Fig. 4). This would be necessary to tag specific synapses recruited by the 182

increased glutamate signaling for future memory consolidation, given that DA seems to be more involved 183

in the tagging process than NE57. For this reason, having two separate release mechanisms might enable 184

more efficient signal processing and storage of new information, since DA released out of the NET would 185

not interfere with the formation of neural representations driven by vesicular NE release. 186

Altogether, these observations concerning the NET’s involvement in DA-dependent potentiation 187

are in conflict with a couple of studies that also measured nisoxetine’s effect on LTP in CA158,59 . The 188

authors of both papers found no difference in LTP when nisoxetine was present. One explanation could 189

be that, in contrast to our methods, these reports did not use any adrenergic receptor antagonists, 190

potentially leading to excess β-adrenergic receptor recruitment and cAMP dependent LTP 191

enhancement42. Although, as mentioned above, our NET knockout assay was also performed in no-drug 192

conditions and produced a massive difference in LTP. A reason for this is not immediately obvious, but 193



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we cannot rule out developmental consequences of NET deletion on normal adrenergic system function. 194

However, a critical observation arising from our experiments is the fact that if LC DA was vesicular in 195

origin, then blocking NET should have the opposite effect, as nisoxetine application should lead to an 196

increase in extracellular DA and thus stronger LTP. 197

In closing, our findings support the idea that the NET and NMDA receptors contribute to DA 198

signaling (Figs. 2&3), and therefore interaction with NE signaling (Fig.4), to regulate attention-guided 199

memory storage in the CA1 region of dorsal hippocampus. One drawback of our methods is that LC fibers 200

were not selectively stimulated. Instead, catecholamine release was elicited by electrical stimulation of all 201

fibers within the range of the stimulating electrode, which could include any other neuromodulatory inputs 202

into CA1 that might interact with the effects of NE and DA (e.g. acetylcholine or serotonin). Also, since we 203

were stimulating with bursts of 100 Hz, this could unnaturally overload LC terminals since their usual 204

maximum firing rate is <20 Hz, leading to DA release out of the NET that would not occur under normal 205

physiological conditions. Future studies may employ specific optogenetic activation of the LC to study this 206

question with greater precision. It will also be necessary to utilize the recently developed genetically 207

encoded fluorescent DA60,61 and NE62 sensors to probe the dynamics of LC catecholamine co-release in 208

greater detail. In conclusion, although our evidence is indirect, it presents a vital first step towards 209

elucidating the complex interplay between glutamate activity and catecholamine release, not only within 210

the hippocampus, but in all LC terminal fields throughout the central nervous system. 211



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Methods 212

Animal Approval 213

All animal procedures performed were approved by the animal care and use committee (IACUC) 214

at the University of Texas Southwestern Medical Center and comply with federal regulations set forth by 215

the National Institutes of Health. 216

Ex vivo slice preparation 217

Coronal slices (300 µm thick) containing dorsal hippocampus were made from male, wild type, 218

C57BL/6J mice (6-12 weeks old) in low-light conditions to prevent photooxidation of catecholamines. 219

Animals were anesthetized under 1.5-2% isoflurane, after which brains were removed and blocked 220

following rapid decapitation. Slices were prepared using a Leica VT1000S vibratome (Wetzlar, Germany) 221

in ice-cold NMDG ringer solution containing (in mM): 5 NaCl, 90 NMDG (N-Methyl-d-Glucosamine), 37.5 222

Na-Pyruvate, 12.5 Na-Lactate, 5 Na-Ascorbate, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 25 Glucose, 10 223

MgSO4.7H20, 0.5 CaCl2.2H20. The pH was set between 7.3 and 7.4 using 12 N HCl, the osmolarity was 224

adjusted as needed to ~315 mOsm using glucose, and the solution was continuously bubbled with 95% 225

O2 and 5% CO2 gas during slicing. Slices were then transferred and maintained for up to 6 hours, while 226

protected from light, at 30 ºC in artificial cerebrospinal fluid containing (aCSF; in mM): 120 NaCl, 3 KCl, 227

1.25 NaH2PO4, 1 MgCl2, 2 CaCl2, 25 NaHCO3, and 11 dextrose continuously bubbled with 95% O2 and 228

5% CO2 gas. 229

Field recordings 230

After at least 1 hour of recovery in aCSF, slices were transferred to a submersion recording 231

chamber and perfused with aCSF at a rate of 2-3 ml/min at 31-32 ºC. Extracellular voltage recordings 232

from the stratum radiatum field of dorsal CA1 were acquired using a borosilicate glass electrode (1-2 MΩ, 233

Sutter Instrument (Novato, CA)) filled with normal aCSF. A bipolar stimulating electrode (FHC, Inc. 234

(Bowdoin, ME)) was also placed in the stratum radiatum of CA1 within ~400 µm of the recording electrode 235

(see Figure 1A), and stimulus strength was controlled with a stimulus isolator unit (World Precision 236

Instruments, Sarasota, FL). Stimulus strength was set to produce a baseline excitatory field postsynaptic 237

potential (fEPSP) slope (Figure 1B) that was ~50% of the slope measured following the first appearance 238

of a population spike. This method led to a typical baseline stimulation current of 20-30 μA, while stimulus 239



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duration was set to 0.2 ms. Schaffer collateral stimulation was given once every 30 seconds and the 240

average of every two consecutive stimuli was taken. For the DA and NE synergy experiments, a stable 15 241

minute control baseline was obtained, followed by another 15 minute baseline with drug washed in. At the 242

end of the 15 minute drug wash, a weak theta-burst tetanus was applied consisting of 5 bursts (given at 5 243

Hz), with each burst containing 5 spikes at 100 Hz (25 total spikes). Baseline stimulation then resumed as 244

described above for 45 minutes. For the NET blockade experiments, the entire experiment was run in the 245

presence of various antagonists (indicated in figures). A 15 minute baseline was obtained, followed by a 246

strong theta-burst tetanus containing 15 bursts (given at 5 Hz), with each burst containing 5 spikes at 100 247

Hz (75 total spikes). Baseline stimulation then resumed as described above for 60 minutes. For 248

Supplementary Data 1, a different LTP stimulation protocol was used that consisted of a 1 second long 249

train of 100 Hz (100 total spikes), without any antagonist application. All experiments were performed in 250

low-light conditions to avoid photooxidation of catecholamines. Data was acquired using a Multiclamp 251

700B amplifier and pCLAMP 10 software (Molecular Devices, San Jose, CA). The signal was low-pass 252

filtered online at 2 kHz using the Multiclamp 700B Commander software, and then digitized at 20 kHz 253

using a Digidata 1440A (Molecular Devices, San Jose, CA). 254

Staining and imaging 255

Dorsal hippocampal sections, 30 µM thick, were cut with a cryostat and stored in 4% PFA in 1X PBS 256

overnight. They were then transferred to a 30% sucrose + 1X PBS solution for cryoprotection. Free 257

floating sections were washed 3x with 1X PBS and treated with a H2O2 solution (PBS + 10% methanol + 258

1.05% H2O2) for at least 1 h. Sections were washed, blocked for 2 h in 10% normal donkey serum + 1X 259

PBS + 1% Triton X-100 (blocking solution) and then were treated overnight at 4ºC with primary antibody 260

diluted in blocking solution containing the following 2 antibodies: mouse monoclonal NET primary 261

antibody, 1:200, Invitrotgen/Thermo Fisher Scientific, (Waltham, MA); rabbit polyclonal NR1 primary 262

antibody, 1:100, Alomone Labs, (Jerusalem, Israel). The following morning, slices were washed and 263

incubated for 2h at RT covered with secondary antibody diluted in blocking solution containing the 264

following 2 antibodies: donkey anti-mouse Alexa Fluor 488, 1:2000, Invitrogen/Thermo Fisher Scientific, 265

(Waltham, MA); donkey anti-rabbit Alexa Fluor 594, 1:1000, Invitrogen/Thermo Fisher Scientific, 266

(Waltham, MA). Sections were next washed, mounted on gelatin covered slides and coverslipped using 267



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PermaFluor (Thermo Fisher Scientific, Waltham, MA) to preserve fluorescence for long-term storage at 268

4ºC. Images were taken on a custom built 2-photon microscope at 20X magnification. 269

Drugs 270

Where indicated, the following drugs were used: (-)-norepinephrine (NE; 20 μM), dopamine (DA; 271

20 μM), prazosin (α1-adrenergic antagonist; 2 μM), propranolol (β-adrenergic inhibitor; 5 μM), SCH 23390 272

(D1-like receptor antagonist; 10 µM), nisoxetine (norepinephrine transporter blocker; 5μM), RS 79948 (α2-273

adrenergic antagonist; 5 μM). All drugs were purchased from Tocris Bioscience (Minneapolis, MN). 274

Statistical analysis 275

All electrophysiological data points are represented as the mean ± SEM. Field recordings were 276

analyzed using two-way repeated measures ANOVAs with time as an independent variable with an 277

assumption for a normal distribution at each averaged data point. Most ANOVAs were run over the last 278

30 minutes of recording after LTP stimulation. However, in Figure 4 an additional ANOVA was run over 279

the 15 minutes that norepinephrine was present in panel A, and ANOVAs were run over the last 15 280

minutes of recording after LTP stimulation since recording only lasted 45 minutes after wLTP stimulation. 281

Also, in Figure 2C another ANOVA was run over the first 30 minutes after LTP. All analyses were 282

performed using GraphPad Prism 7 software (San Diego, CA). 283

284



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Data Availability 285

The data that support the findings of this study are available from the corresponding author upon 286

reasonable request. 287



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62 Feng, J. et al. A Genetically Encoded Fluorescent Sensor for Rapid and Specific In Vivo 458 Detection of Norepinephrine. Neuron 102, 745-761 e748, doi:10.1016/j.neuron.2019.02.037 459 (2019). 460

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Acknowledgements 463

We would like to thank Dr. Marc G. Caron for his generous gift of norepinephrine transporter knockout 464 mice. We would also like to thank To Thai for his technical assistance. 465

Author contributions 466

A.S. designed the experiments, performed the experiments, analyzed data, and wrote the paper. 467

R.W.G. designed the experiments and edited the paper. 468

Additional information 469

The authors declare no competing interests. 470

471



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Figure 1. Establishment of weak and strong long-term potentiation (LTP) protocols 472

A, Diagram of a hippocampal slice with electrodes in place. The stimulating electrode (left) is placed in 473

contact with Schaffer collateral axons from the CA3 region about 400 µm from the recording electrode. 474

The recording electrode (right) measures the extracellular field excitatory postsynaptic potential (fEPSP) 475

in stratum radiatum dendrites of CA1. B, Example fEPSP from CA1. Data is taken as the initial slope of 476

the voltage trace as shown in red. Scale bars represent the 0.5 millivolt amplitude and two millisecond 477

duration in all of the following figures. C, Weak (wLTP, n=8) and strong (strLTP, n=7) Schaffer collateral 478

thetaLTP stimulation protocols (see methods for more details). D, Weak versus strong thetaLTP time 479

course. The black arrow represents the moment that either LTP stimulation was given after a 15 minute 480

baseline. Insets are representative traces before and after each stimulation protocol. The solid lines 481

represent an average of baseline traces from 0-15 minutes before LTP stimulation, while dotted lines 482

represent an average of traces from the last 5 minutes of the recording after LTP stimulation. E, strLTP 483

(open circles, n=7) is not blocked by the addition of prazosin and propranolol to the bath (closed circles, 484

n=6), F(1, 17) = 0.06472, p=0.8022, ‘n.s.’ stands for ‘not significant’. All data points are represented as 485

mean +/- SEM. Tests for significance were done using a two-way repeated measures ANOVA over the 486

last 30 minutes of strLTP. 487



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Figure 2. The norepinephrine transporter (NET) contributes to dopamine-dependent long-term 488 potentiation 489

A, The previously established strong LTP protocol (strLTP, black arrow) was not blocked by application of 490

antagonists for β- and α1-adrenergic receptors, propranolol and prazosin, respectively (black circles, n=6, 491

see Fig. 1E). However, application of SCH 23390, a dopamine D1-like receptor antagonist, along with β- 492

and α1 blockers was enough to significantly reduce the last 30 minutes of LTP (red circles, n=6), F(1, 10) 493

= 9.265, p=0.0124. B, Similar to A, but the D1/5 receptor antagonist was replaced with the NET blocker 494

nisoxetine (green squares, n=6), which was sufficient to attenuate the dopamine-dependent LTP (black 495

circles, n=6), F(1, 10) = 5.028, p=0.0488. C, Blockade of ALL adrenergic receptors (by adding an α2 496

autoreceptor antagonist) selectively increased the first 30 minutes of LTP (blue triangles, n=6) compared 497

to β- and α1 blockers alone (black circles, n=12), F(1, 16) = 4.963, p=0.0406. D, Even with all adrenergic 498

receptors blocked (blue triangles, n=6) the application of nisoxetine was still able to significantly reduce 499

LTP (green triangles, n=6), F(1, 10) = 5.521, p=0.0407. All data points are represented as mean +/- SEM. 500

Tests for significance were done using a two-way repeated measures ANOVA over the last 30 minutes of 501

strLTP, or during the first 30 minutes as shown in panel C. Asterisks represent p-values <0.05, while ‘n.s.’ 502

stands for ‘not significant’. 503



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504

Figure 3. Knocking out NMDA receptors from catecholamine neurons reduces the magnitude of 505

dopamine-dependent LTP in dorsal hippocampus 506

A, The same strong LTP protocol used previously (strLTP, black arrow) was administered in slices from 507

Cre(-) control mice (black circles, n=6) and NMDA-NR1 subunit knockout mice (open circles, n=6). All 508

data points are represented as mean +/- SEM. Tests for significance were done using a two-way 509

repeated measures ANOVA over the last 30 minutes of strLTP. Double asterisk represents a significant 510

difference <0.01, F(1, 10) = 13.24, p=0.0046. 511



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Figure 4. Weak LTP (wLTP) is enhanced by 512

dopamine and norepinephrine together but 513

not by either of them alone 514

A, wLTP (black arrow, black circles, n=8) is 515

not enhanced with bath application of 20 μM 516

norepinephrine alone (blue squares, 61-76 517

min, n=6), F(1, 12) = 0.05005, p=0.8267. 518

Instead, the application of norepinephrine after 519

a 15 minute baseline significantly reduced the 520

size of the baseline field potential slope (blue 521

squares, 16-30 min, n=6), F(1, 12) = 18.87, 522

p=0.0010. B, Bath application of 20 μM 523

dopamine (red diamonds, n=8) was also 524

unable to enhance wLTP, but did not show a 525

similar decrease of baseline slope, F(1, 14) = 526

0.1202, p=0.7339. C, Application of 20 μM 527

norepinephrine and 20 μM dopamine together 528

(green triangles, n=7) produces a significant 529

increase of wLTP, F(1, 13) = 9.318, p=0.0080. 530

All data points are represented as mean +/- 531

SEM. Tests for significance were done using a 532

two-way repeated measures ANOVA over the 533

last 15 minutes of wLTP, or during 15 minutes 534

of drug application as shown in panel A. 535

Asterisks represent p-values < 0.05, double 536

asterisks represent p-values < 0.01, while ‘n.s.’ 537

stands for ‘not significant’. 538

539



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540 541

Supplementary Figure 1. Knocking out the norepinephrine transporter (NET) reduces the 542

magnitude of LTP in dorsal hippocampus 543

A different LTP protocol was administered (100 Hz for 1 second) without the addition of any adrenergic 544 receptor antagonists in slices from Cre(-) control mice (black circles, n=12) and NET knockout mice (open 545 circles, n=4). All data points are represented as mean +/- SEM. Tests for significance were done using a 546 two-way repeated measures ANOVA over the last 30 minutes of 100 Hz LTP. Double asterisk represents 547 p<0.01, F(1, 14) = 15.59, p=0.0015. 548

549



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550

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Supplementary Figure 2. Colocalization of the norepinephrine transporter and presynaptic NMDA 566 receptors in dorsal CA1 567

A,B,C, Representative immunnostaining image of LC fibers in the CA1 region of the dorsal hippocampus 568 showing co-localization (C) of the norepinephrine transporter (NET, green, A) and presynaptic NMDA 569 receptors (NR1, magenta, B) on LC terminals. 570



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